Electrofusion microelectrode

ABSTRACT

The present invention is directed to an electrofusion microelectrode used in the alignment, manipulation, fusion, or electroporation of cells. This device is particularly useful for transplantation of cells and cellular components.

BACKGROUND OF THE INVENTION

[0001] Electrofusion and electroporation of cells involves applicationof an electrical current to cells. In many instances, cells are alignedprior to applying a direct electrical current. Alignment may be donemanually, for example, by aspiration or vacuum suction. Alignment mayalso be performed by applying an alternate electrical current. Whenalignment is done by applying alternate current, cell survival isdrastically reduced. The present invention provides a tool having thedual capacity to manually align cells and deliver direct current tocells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0002]FIG. 1 is an illustration of one embodiment of the electrofusionmicroelectrode. In this embodiment, the first end of the tube is sealed.

[0003]FIG. 2 is an illustration of another embodiment of theelectrofusion microelectrode. In this embodiment, the first end of thetube is open.

[0004]FIG. 3 is an illustration depicting the electrofusionmicroelectrode connected to a power source via an electrode clip. Inthis illustration, the microfilament protrudes from the tube at thedistal end and is bent or looped. The electrode clip is clamped on thedistal end of the tube and also contacts the bent portion or loop of theconducting filament. Suction means via a pipette holder is also depictedin this figure and may be connected to the distal end of theelectrofusion microelectrode.

DESCRIPTION OF THE INVENTION

[0005] The present invention is directed to an electrofusionmicroelectrode which may be used in the alignment, manipulation, fusionor electroporation of cells including the transplantation of cells andcellular components. The electrofusion microelectrode comprises a tubeencasing a filament which is an electric conductor. As used herein,“tube” is meant to encompass any hollow casing and may have any type ofgeometrical conformation. Thus, if desired, the walls of the tube may beangled. In a preferred embodiment, the tube is cylindrical. In an evenmore preferred embodiment, the tube is shaped as a holding pipette.

[0006] The tube as well as the conducting filament has both a medial anddistal end. As used herein, the “medial end” of the tube or conductingfilament is the end which contacts the cells and/or cellular components.The “distal end” of the tube or conducting filament is furthest awayfrom the cells and/or cellular components and nearer a direct currentpower source.

[0007] The conducting filament may comprise any known conductor such asa metal, metal alloy or mixture of metals and/or metal alloys. Certaincarbon allotropes may also be used to form the conducting filament.Examples of metal conductors which may be used as a conducting filamentinclude but are not limited to, aluminum, copper, silver, gold,titanium, platinum, and tungsten. An example of a carbon allotrope whichmay be used as a conducting filament in the electrofusion microelectrodeis graphite. In a preferred embodiment, the metal filament is made oftungsten or tungsten alloy.

[0008] In one embodiment of the invention, one end of the filament isflattened at the tip of one end of the tube and the tube is sealed atthis end (medial end). In an alternative embodiment, the electrofusionmicroelectrode has an internal opening surrounding the filament. Theinternal opening is useful to allow aspiration or vacuum suctioning ofcells such as used with a standard holding pipette. In this embodiment,the end of the tube which is in contact with the cells or cellularcomponents, i.e., the medial end, is open. In FIG. 1, the first (medial)end of the tube where the first (medial) end of the filament protrudesis sealed. FIG. 2 shows an alternative embodiment where the first end ofthe tube is open.

[0009] The tube portion of the electrofusion microelectrode may be madeof any number of materials such as glass, plastic, PVC, ceramic, metal,etc. In a preferred embodiment, the tube portion is made of glass. In amore preferred embodiment, the tube portion is made from a borosilicateglass capillary tube, pulled and forged as a holding pipette.

[0010] The length and diameter of the electrofusion microelectrode mayvary according to the type of cells and type of manipulation for whichthe tool is used. For example, when used for nuclear transplantation ofmammalian cells, a tube diameter in the range of from about 15 to about25 μm is useful. When used for mammalian cell fusion, a tube diameter inthe range of from about 60 to about 100 μm may be used. Thus, in oneembodiment, the outer diameter of the tube may be about 0.97 mm whilethe inner diameter of the tube may be about 0.69 mm. In this embodiment,the tube is quite thin walled, having a thickness of only about 0.28 mm.The diameter of the conducting filament may be anywhere in the range offrom about 7 to about 20 μm. The distal end of the conducing filament ispreferably thicker than the medial end so that connection to a powersource is conveniently achieved.

[0011] The length of the microelectrode can of course, vary. A length ofabout 78 mm is convenient for most manipulations. Typically, there is abend in the tube approximately 1 mm or so from the medial end.

[0012] With reference to FIG. 1, one embodiment of the invention isillustrated therein and it will be seen to include an electrode mainbody and an electrode tip. As illustrated in FIG. 1, a conductorfilament extends throughout the tube (electrode main body). Both thetube and the filament have a first (medial) and a second (distal) end. Afirst (medial) end of a filament protrudes through a first (medial) endof the tube and is flattened at the tip of the first (medial) end of thetube. In FIG. 1, the medial ends make up the electrode tip. A second(distal) end of the filament protrudes through a second (distal) end ofthe tube and is configured to both allow the filament to remainrelatively fixed within the tube and to allow connection to a powersource. The medial end of the tube may be open, or closed (sealed).

[0013] There are many possible configurations for the distal, protrudingend of the filament. In one embodiment, the distal end of the filamentmay be bent or looped towards the outer wall of the tube or wrappedaround the outer wall of the tube in order to have the filament remainrelatively fixed within the tube. Conveniently, an electrode clip or thelike may be clamped around the tube as well as the looped, bent, orwrapped portion of the distal end of the filament. This embodiment ofthe invention is depicted in FIG. 3.

[0014] In another embodiment of the invention, the medial end of theconducting filament does not protrude from the medial end of the tube.The inner walls of the tube are painted with a liquid form of anelectric conductor from a place where the filament no longer extends tothe medial end of the tube and the paint extends to the outside(lateral) edge of the medial end of the tube. In this embodiment, thedistal ends of the filament and tube are as described above. Again, themedial end of the tube may be open or closed (sealed).

[0015] In yet another embodiment of the invention, rather than using aconducting filament, the inner portion of the tube is painted with aliquid form of an electric conductor. Examples include liquid aluminum,copper, silver, gold, titanium, platinum, tungsten, and alloys andmixtures thereof. Thus, at least a portion of the inner walls arepainted with a liquid electric conductor and the painted area extendscontinually from the medial end of the tube to the distal end of thetube. The medial end of the tube may be opened or sealed. The liquidconductor is also painted on at least a portion of the outer (lateral)edge of both the medial and distal ends of the tube. Further, a portionof the liquid conductor is applied to the outside wall of the tube atthe distal end so that connection to a direct current power source maybe achieved. For example, an electrode clip may be clamped to the tube,contacting that portion of the distal end of the outer wall of the tubewhich is painted with the liquid conductor.

[0016] The electrofusion microelectrode is preferably mounted on a toolholder where it can be controlled by a micromanipulator. Preferably, themicromanipulator is used under inverted microscopy. Examples ofmicromanipulators which may be used with the subject electrofusionmicroelectrode include but are not limited to, the MM188 and MM109manufactured by Narishigie Co., LTD, Tokyo, Japan. Preferably, theelectrofusion microelectrode is used as a set of two: the distal end ofthe conducting filament of one electrofusion microelectrode beingconnected to the positive terminal of a direct current power source, andthe distal end of the conducting filament of a second subjectelectrofusion microelectrode being connected to the negative terminal ofa direct current power source. The power source should be able todeliver at least 1 kilovolt per centimeter, direct current. Examples ofpower sources that may be used with the subject microelectrode includethe BTX Electro Cell Manipulator 200 or 2001 (BTX Inc., San Diego,Calif.).

[0017] The subject tool(s) may be used to perform the techniques ofelectrofusion/electroporation by manually aligning and/or ormicromanipulating the cells using microelectrode motion. Alternatively,if the medial end of the tool(s) is open, cells may be manually alignedusing aspiration or vacuum suction. Of course, a combination ofmicroelectrode motion and aspiration or suction may be used tomicromanipulate and/or align cells. After aligning cells, direct currentmay be applied via the subject microelectrode(s). Since cells arealigned manually, the use of alternate current for alignment is avoided,significantly improving cell survival. Since cell survival isdrastically improved, much lower cell numbers may be used in eachmanipulation.

[0018] The present invention therefore provides methods of manipulatingcells using the subject electrofusion microelectrode. Such methodsinclude for example, cell transplantation, electrofusion of cells,electroporation of cells, and nuclear transplantation. Thus, the presentinvention provides a method of transplanting mammalian cells whichcomprises micromanipulating the cells with two electrofusionmicroelectrodes and delivering a direct current to the manipulatedcells. The subject electrofusion microelectrodes for use in the methodof transplantation of mammalian cells, may have any of the alternateembodiments hereinbefore described.

[0019] The present invention also provides a method of electrofusion ofcells. The method comprises aligning cells between two electrofusionmicroelectrodes and delivering a direct current to the aligned cells.Again, the subject electrofusion microelectrodes for use in the methodof electrofusion of cells, may have any of the alternate embodimentshereinbefore described.

[0020] Also provided by the present invention is a method ofelectroporation of cells. The method comprises manipulating cells withtwo electrofusion microelectrodes and delivering a direct current to themanipulated cells. The subject electrofusion microelectrodes for use inthe method of electroporation may have any of the alternate embodimentshereinbefore described.

[0021] A method of nuclear transplantation is also provided by thepresent invention. The method comprises removing a nucleus from a firstoocyte and transplanting the nucleus into the perivitelline space of asecond, previously enucleated oocyte, and then integrating thetransplanted nucleus of the first oocyte with the cytoplasm of thesecond oocyte. Transplantation and integration is performed using thesubject electrofusion microelectrodes and integration is achieved bydelivering a direct current to the nucleus and cytoplasm. The subjectelectrofusion microelectrodes for use in the method of nucleartransplantation may have any of the alternate embodiments hereinbeforedescribed.

EXAMPLE I

[0022] A capillary tube, 78 mm in length, and having an outer diameterof 0.97 mm and an inner diameter of 0.69 mm (Drummond Scientific,Boomall, Pa.), is pulled on a horizontal microelectrode puller(micropuller) (Campden Inc., LTD., London) approximately 60 to 100 μm ata location of 10-15 mm from one end (medial end). The tube is cut andfine polished on a microforge (Narishige Co., LTD, Tokyo, Japan) toobtain a final outer diameter of 60 μm and an inner diameter of 20 μm. Aplatinum filament having a thickness of about 20 to 40 μm (availablefrom a fine jeweler) is inserted into the distal end of the pipetteunder a sterile microscope with a magnification of 6-15× or a magnifyinglens of at least 6×. The medial end of the conducting filament is placedflush against the tip of the medial end of the pipette. The distal endof the filament is of a length longer than the pipette so that that itexits the distal end of the pipette by a length of at least 10 mm. Thisportion of the filament which exits the distal end of the pipette isbent towards the outside wall of the distal end of the pipette, making abend or a loop to secure the filament in place within the pipette and toallow connection to a power source by means of an electrode clip. Anelectrode clip may be attached to the tube, ensuring that contact withthe protruding portion of the distal end of the filament is made (FIG.3). The electrode clip may be connected to a direct current power sourcesuch as the BTX Electro Cell Manipulator 200 or 2001 (San Diego,Calif.).

EXAMPLE II Nuclear Transplantation for Immature Mammalian Oocytes

[0023] Germinal vesicle (GV) stage oocytes are retrieved by puncturingfollicles of unstimulated ovaries of B6D2F1 female mice. A karyoplast isthen removed by micromanipulation using one or more of the subjectelectrofusion microelctrodes in a medium supplemented with cytochalasinB. One karyoplast is subsequently introduced into the perivitellinespace of a previously enucleated immature oocyte. Each grafted oocyte isthen positioned between two of the subject electrofusion microelectrodesand exposed to a single or double 1.0 kV/cm, 50-99 μm direct currentfusion pulse(s). Thirty to 60 minutes later, the oocytes are examinedfor sign of fusion. The restored oocytes are then placed in culture andassessed for maturation. Oocytes which have extruded a first polar bodymay be fixed and stained with Giemsa for chromosome analysis. Ascontrols, approximately one third of oocytes are not subjected to anymanipulation, but are merely cultured in the same media and exposed tosame reagents.

EXAMPLE III Germinal Vesicle Transplantation

[0024] Germinal vesicle (GV) stage oocytes are retrieved by puncturingfollicles of unstimulated ovaries of B6D2F1 female mice. Metaphase II(MII) oocytes are collected 15 hours after hCG injection of PMSGstimulated females. Karyoplasts are then removed from GV oocytes usingone or more subject electrofusion microelectrodes, in a mediumsupplemented with cytochalsin B. MII oocytes are enucleated by removingthe “hub” area where the metaphase spindle is located, together with thefirst polar body using one or more of the subject electrofusionmicroelectrodes. A GV karyoplast is subsequently introduced into theperivitelline space of either a previously enucleated immature (GV) or amature (MII) oocyte. Each of these manipulated oocytes is thenpositioned between two of the subject electrofusion microelectrodes andexposed to a single or double 1.0 kV/cm, 50-99 μm direct current fusionpulse(s) for electrofusion. The oocytes that show signs of fusion 30 to60 minutes later are then placed in culture for 12 hours, to allownuclear maturation. Oocytes which extrude the first polar body may befixed and stained with Giemsa for chromosome analysis.

What is claimed:
 1. An electrofusion microelectrode for manipulatingcells or cellular components which comprises a conducting filamentencased in a tube, wherein a first (medial) end of the filamentprotrudes from, and is flattened against, a first (medial) end of thetube, and wherein a second (distal) end of the filament protrudes from asecond (distal) end of the tube and wherein the second (distal) end ofthe filament is configured to allow the filament to remain relativelyfixed within the tube and to allow connection to a direct current powersource.
 2. An electrofusion microelectrode which comprises a tube,wherein at least a portion of the inner walls are painted with a liquidelectric conductor and wherein the painted electric conductor extendscontinually from a first (medial) end of the tube to a second (distal)end of the tube, wherein the liquid electric conductor is furtherpainted on a least a portion of the outer (lateral) edge of both thefirst (medial) and second (distal) ends of the tube, wherein an area onthe outer wall of the tube at the second (distal) end is painted withthe electric conductor and wherein the painted area of the outer wall ofthe tube at the second (distal) end meets the painted area of the outer(lateral) edge of the second (distal) end of the tube, and wherein thedistal end of the tube is connectable to a direct current power source.3. An electrofusion microelectrode which comprises a conducting filamentencased in a tube, wherein a first (medial) end of the filament extendstoward a first (medial) end of the tube, and wherein a second (distal)end of the conducting filament protrudes from a second (distal) end ofthe tube, wherein at least a portion of the inner walls near the first(medial) end of the tube is painted with an electric conductor in anarea where the conducting filament does not extend, and wherein thesecond (distal) end of the conducting filament is configured to allowthe filament to remain fixed within the tube and to allow connection toa direct current power source.
 4. The electrofusion microelectrode ofany of claims 1-3 wherein the tube is shaped as a holding pipette. 5.The electrofusion microelectrode of any of claims 1-3 wherein the first(medial) end of the tube is sealed.
 6. The electrofusion microelectrodeof any of claims 1-3 wherein the first (medial) end of the tube is open.7. The electrofusion microelectrode of any of claims 1-3 wherein thetube is made of plastic, PVC, ceramic, or metal.
 8. The electrofusionmicroelectrode of any of claims 1-3 wherein the tube is made of glass.9. The electrofusion microelectrode of claims 1 or 3 wherein theconducting filament is made of a metal, metal alloy, or mixture ofmetals.
 10. The electrofusion microelectrode of claim 9 wherein themetal or metal alloy is at least one of aluminum, copper, silver, gold,titanium, platinum, or tungsten.
 11. The electrofusion microelectrode ofany of claims 1-3 wherein the second (distal) end of the tube isconnectable to a vacuum or hand held aspirator.
 12. The electrofusionmicroelectrode of claim 11 wherein the hand held aspirator is a pipetteholder.
 13. The electrofusion microelectrode of any of claims 1-3wherein the electrofusion microelectrode is mounted on a tool holder.14. The electrofusion microelectrode of claim 13 wherein the tool holderis controlled by a micromanipulator.
 15. The electrofusionmicroelectrode of claim 14 wherein the micromanipulator is used underinverted microscopy.
 16. The electrofusion microelectrode of claims 1 or3, wherein the second (distal) end of the conducting filament isconfigured by being bent or looped towards the outer wall of the tube orbeing wrapped around the outer wall of the tube.
 17. A method oftransplanting mammalian cells which comprises micromanipulating thecells with two electrofusion microelectrodes and delivering a directcurrent to the manipulated cells wherein the electrofusionmicroelectrodes comprise the electrofusion microelectrode of any one ofclaims 1-3.
 18. A method of electrofusion of cells which comprisesaligning cells between two electrofusion microelectrodes and deliveringa direct current to the aligned cells, wherein the electrofusionmicroelectrodes comprise the electrofusion microelectrode of any one ofclaims 1-3.
 19. A method of electroporation of cells which comprisesmanipulating cells with two electrofusion microelectrodes and deliveringa direct current to the manipulated cells, wherein the electrofusionmicroelectrodes comprises the electrofusion microelectrode of any one ofclaims 1-3.
 20. A method of nuclear transplantation which comprisesremoving a nucleus from a first oocyte and transplanting the nucleusinto the perivitelline space of a second, previously enucleated oocyte,integrating the transplanted nucleus of the first oocyte with thecytoplasm of the second oocyte, wherein the transplantation andintegration is performed using two electrofusion microelectrodes,wherein the integration is performed by delivering a direct current tothe nucleus and cytoplasm, and wherein the electrofusion microelectrodescomprise the electrofusion microelectrode of any one of claims 1-3.